Registration Dossier

Administrative data

Description of key information

The SI obtained for chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 at all the tested concentrations showed a less than three-fold increase over the vehicle control value. Therefore, chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 did not demonstrate skin sensitisation potential in the local lymph node assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation: March 14, 2018 and Study Completion date : April 20, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Test System : Mice
Species (strain): Mus musculus (CBA/J)
Animal Source: Animal Breeding Facility, Jai Research Foundation
Number of Animals Used: 8 (2 mice/group) for preliminary assay and
25 (5 mice/group) for main study
Sex:Female (nulliparous and non-pregnant)
Initial Body Weight (g): On Day 0 (Main study): Minimum: 19.0, Maximum: 25.1
Age (Main study): 10 to 11 weeks old at the initiation of treatment
Acclimatisation period: At least 6 days
Veterinary health check: Before and during acclimatisation period
Feed: Teklad Certified Global High Fiber Rat/Mice Feed manufactured by Envigo, USA, was provided ad libitum.
Water: UV sterilised water (Reverse Osmosis water filtration system) was provided ad libitum.

ENVIRONMENTAL CONDITIONS
Caging: Solid floor polypropylene mice cages (size: 290 mm x 220 mm x 140 mm). Each cage is fitted with a top grill having provision for keeping rodent pellet feed and water bottles. The bottom of the cages is layered with clean sterilized rice husk as the bedding material. Animals were group-housed during acclimatisation. On the days of test item application (Days 0, 1 and 2), the animals were housed in individual cages. From day 3 the animals were group-housed 5 mice/cage. On day 5 post administration of the radiolabelled material the animals were transferred to the metabolic cages. Tunnels were provided as enrichment material.
Water Bottle : Each cage was supplied with a polypropylene water bottle (capacity 300 mL) with a stainless steel nozzle.
Room Sanitation : Each day, floor of experimental room was swept and all work tops and floor were mopped with a disinfectant solution (Dettol 2.5%).
Enrichment Material : Tunnel

Animal Room : BMR – 30 [On day 5, all animals were shifted to Room N° 11/A]
Temperature : 20 to 23 °C
Relative Humidity : 57 to 67%
Photoperiod : The photoperiod was 12 h artificial light and 12 h darkness, light hours being 06:00 – 18:00 h (photoperiod maintained through automatic timer).
Air Changes Rate : Minimum 15 air changes/hour
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test substance: 1%, 5% and 10% (w/w)
No. of animals per dose:
5 animals per dose
Details on study design:
PRE-SCREEN TESTS:
-Compound solubility: The test item formed good suspension in acetone:olive oil (4:1) (AOO) up to the dose concentration of 10% (w/v).
-Irritation: Clinical observations including scoring of irritation were recorded daily during the experiment.
- Systemic toxicity: Clinical observations including systemic clinical signs were recorded daily during the experiment
- Ear thickness measurements: Ear thickness was measured on days 0, 2 and 5.
-Body weight: was recorded on days 0 and 5.
- Erythema scores: Erythema and eschar formation Value
No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay – Mice (CBA/J); OECD 429
- Criteria used to consider a positive response: Stimulation indices (SI) for the 1%, 5% and 10% (w/v) in acetone:olive oil (4:1) (AOO) treated groups were 0.97, 1.33 and 2.01, respectively.
A positive response for HCA (SI = 5.45) confirmed the reliability of the test procedure.
The SI obtained for the test substance at all tested concentrations showed a less than three-fold increase over the vehicle control value, which does not demonstrate sensitisation potential in the local lymph node assay.

TREATMENT PREPARATION AND ADMINISTRATION:
Treatment: Three groups (G2 to G4) were treated topically for three consecutive days (days 0, 1 and 2) on the dorsal surface of both ears (25 micro L/ear) using a calibrated micropipette at concentrations of 1%, 5% and 10% (w/v) test substance in acetone:olive oil (4:1) (AOO), respectively. Mice from vehicle control group (G1) and positive control group (G5) were handled in the same manner but received 25 microL/ear of vehicle [acetone:olive oil (4:1) (AOO)] and 25% a-Hexylcinnamaldehyde (v/v) in vehicle (AOO), respectively.
-Administration: On day 5, all mice from the vehicle control, positive control and all the treatment groups were injected with 250 µL of sterile phosphate buffered saline (PBS) containing 20.63 µCi of 3H-methyl thymidine via the tail vein.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI of 5.45 obtained for the positive control, Hexylcinnamaldehyde, showed greater than a three-fold increase over the vehicle control value indicating a positive response in agreement with the historical control for this known weak sensitiser. This confirmed the reliability of this test procedure.
Key result
Parameter:
SI
Value:
0.97
Test group / Remarks:
1%
Remarks on result:
other: Not classified as a skin sensitiser
Key result
Parameter:
SI
Value:
1.33
Test group / Remarks:
5%
Remarks on result:
other: Not classified as a skin sensitiser
Key result
Parameter:
SI
Value:
2.01
Test group / Remarks:
10%
Remarks on result:
other: Not classified as a skin sensitiser
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA :
Proliferative responses in the draining lymph nodes were monitored by measuring the incorporation of 3H-methyl thymidine. These analyses revealed group mean DPM values of 875.20, 852.20, 1165.80 and 1759.60 for the vehicle control (AOO), 1%, 5% and 10% (w/v) test substance in acetone:olive oil (4:1) (AOO), respectively.

DETAILS ON STIMULATION INDEX CALCULATION:
Stimulation Index (SI) values calculated for groups treated with the test substance were found to be 0.97, 1.33 and 2.01 at the dose concentrations of 1%, 5% and 10% (w/v) the test substance in acetone:olive oil (4:1) (AOO), respectively

CLINICAL OBSERVATIONS:
No clinical signs were observed in any mice from the vehicle control, positive control and treated groups at 1%, 5% and 10% (w/v) the test substance in acetone:olive oil (4:1) (AOO).
No erythema was observed in any treated mice at 1%, 5% and 10% (w/v) the test substance in acetone:olive oil (4:1) (AOO) on day 0 to day 5.

BODY WEIGHTS:
The mean body weight of positive control and the test substance treated mice was comparable to that of the vehicle control group.
Interpretation of results:
other: Not classified as a skin sensitiser
Remarks:
Not classified as a skin sensitiser
Conclusions:
The SI obtained for chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 at all the tested concentrations showed a less than three-fold increase over the vehicle control value. Therefore, chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 did not demonstrate skin sensitisation potential in the local lymph node assay.
Executive summary:

A preliminary assay was conducted to identify the appropriate test concentrations for the Local Lymph Node Assay for main study. Thechlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18did not induce irritation either in terms of erythema or increase of ear thickness.

In the main assay, five groups of mice comprising 5 females per group were selected. Based on the results of the preliminary assay, three groups were treated at dose concentrations of 1%, 5% and 10% (w/v)chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18inacetone:olive oil (4:1) (AOO) for three consecutive days (days 0, 1 and 2) on the dorsum of both ears (25mL per ear). In addition, one group served as the vehicle control and was treated with acetone:olive oil (4:1) (AOO) and another group served as the positive control and was treated with 25% (v/v) HCA (a-hexylcinnamaldehyde) in acetone:olive oil (4:1) (AOO).

Animals were observed for local irritation and clinical reactions daily. Body weight was conducted at the beginning and at the end of treatment. On day 5, the uptake of intravenously injected3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post-administration) to assess the lymph node proliferative response.

There were no indications of local irritation or systemic toxicity in chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 treated animals. In all mice treated with 25% HCA (v/v), a local reaction consisting of very slight erythema (score of 1) was observed from days 1 to 4. Group mean body weight of treated animals and positive control group animals were comparable with the mean body weight of the vehicle control group.

Stimulation indices (SI) for the 1%, 5% and 10% (w/v) inacetone:olive oil (4:1) (AOO)treated groups were 0.97, 1.33 and 2.01, respectively.

The SI obtained for chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18at all tested concentrations showed a less than three-fold increase over the vehicle control value, which does not demonstrate sensitisation potential in the local lymph node assay.

Based on the results of this study, an indication of the classification for chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 is: Globally Harmonized System of Classification and Labeling of Chemicals (GHS 2017) : Not classified as a skin sensitiser

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

The SI obtained for chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 at all the tested concentrations showed a less than three-fold increase over the vehicle control value. Therefore, chlorinated reaction product of Vat Blue 20 (violanthrone A, violanthrone B and iso-violanthrone)-earlier known as Vat Blue 18 did not demonstrate skin sensitisation potential in the local lymph node assay.